Bacillus velezensis and application thereof in preparation of water-soluble microbial agent

Abstract

The invention relates to bacillus velezensis and application thereof in preparation of a water-soluble microbial agent, and belongs to the technical field of microorganisms. The bacillus velezensis has the effects of producing exopolysaccharides, promoting the formation of soil aggregates, degrading abamectin, hydrolyzing protein, producing indoleacetic acid, promoting the growth of tomato plants and promoting the enzyme activity of tomato rhizosphere soil enzyme. The fertilizer not only has the effects of improving the soil aggregate structure and promoting the growth of tomato plants, but also can accelerate the temperature rise of the abamectin fermentation waste residue compost, prolong the high-temperature period and shorten the compost period, and is beneficial to promoting the harmless treatment and resource utilization of the abamectin fermentation waste residue.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Bacillus Velezii and application thereof in preparing a water-soluble microbial agent. Background Art

[0002] With the rapid development of biotechnology and water-fertilizer integration technology, the application of microbial agents is becoming more and more extensive. Water-soluble microbial agents integrate functional bacteria and soluble organic matter, can be drip-irrigated or flushed, and have the characteristics of soil improvement, promoting crop growth, and enhancing crop disease resistance. At present, the development of water-soluble microbial agents that integrate functional bacteria and soluble organic matter and are suitable for water-fertilizer integration has become a research hotspot.

[0003] At present, the main raw materials for the production of water-soluble microbial agents used for drip irrigation or flushing are water-soluble organic raw materials, such as fulvic acid, amino acids, molasses and wood vinegar, etc. However, due to the limited sources of the above raw materials and high prices, the large-scale promotion and application of water-soluble microbial agents are limited. The use of fermentation or enzymatic hydrolysis can convert water-insoluble organic raw materials into water-soluble organic raw materials and apply them to the production of water-soluble microbial agents, which has broad development potential, but there are relatively few related studies at present.

[0004] Abamectin fermentation waste residue is a by-product of the antibiotic fermentation industry. The waste residue is rich in proteins, amino acids, carbohydrates, trace elements, and residual mycelium. It is nutritious and inexpensive, and is an ideal raw material for the production of water-soluble microbial agents by fermentation or enzymatic methods. However, the residual avermectin in the avermectin fermentation waste residue poses a huge challenge to its comprehensive utilization. During the enzymatic hydrolysis process, since avermectin cannot be enzymatically hydrolyzed, it will remain in the hydrolyzate, posing a potential risk; during the fermentation hydrolysis process, the inhibitory effect of avermectin on the fermentation microorganism will cause the fermentation process to be blocked, and if avermectin is not effectively degraded by the microorganism, it will also remain in the hydrolyzate. Therefore, when utilizing avermectin fermentation waste residue, it is necessary to select microorganisms that can effectively degrade avermectin for treatment. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a strain of Bacillus Velezii and application thereof in the preparation of a water-soluble microbial agent.

[0006] The technical solution of the present invention is as follows: A strain of Bacillus velez Bacillus velezensis ) NKBV-01, deposited on November 6, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32500.

[0007] The 16S rDNA sequence of the Bacillus Velezii NKBV-01 is shown in SEQ ID NO.1.

[0008] The Velez Bacillus NKBV-01 is used in producing extracellular polysaccharides, promoting soil aggregate formation, degrading avermectin, hydrolyzing protein, producing indoleacetic acid, promoting tomato plant growth, and promoting the enzyme activity of tomato rhizosphere soil enzymes.

[0009] Preferably, the tomato rhizosphere soil enzymes include urease, catalase and sucrase.

[0010] A water-soluble microbial agent comprises the Bacillus Velez subtilis NKBV-01.

[0011] Preferably, the water-soluble microbial agent also includes low-value fish hydrolysate dry powder and Kathon.

[0012] Further preferably, the water-soluble microbial agent comprises the following components: 75-85 parts by weight of Bacillus Velez NKBV-01 fermentation broth, 14.85-24.95 parts by weight of low-value fish enzymatic hydrolysate dry powder, and 0.05-0.15 parts by weight of Kathon.

[0013] More preferably, the water-soluble microbial agent comprises the following components: 80 parts by weight of Bacillus Velez NKBV-01 fermentation broth, 19.9 parts by weight of low-value fish enzymatic hydrolysate dry powder, and 0.1 parts by weight of Kathon.

[0014] The preparation method of the water-soluble microbial agent comprises the following steps: adding low-value fish enzymatic hydrolysate dry powder and kason to the fermentation liquid of Bacillus Velez NKBV-01, and mixing them evenly to obtain the water-soluble microbial agent.

[0015] Preferably, the preparation method of the low-value fish hydrolysate dry powder comprises the following steps: using low-value fish meal as a substrate, preparing a slurry with a substrate mass percentage of 8% to 12%, heating the slurry at 80 to 90°C for 10 to 20 minutes to achieve protein denaturation; after the slurry is cooled, adding a composite protease at a ratio of 1% to 3% of the substrate mass percentage, and then enzymolyzing for 3 to 5 hours at 50 to 55°C; after the enzymolysis is completed, heating to 80 to 90°C and maintaining for 10 to 20 minutes to inactivate the enzyme, and then reducing the temperature to below 55°C; performing solid-liquid separation to obtain a filtrate, concentrating the filtrate to a solid mass percentage of ≥25%, and spray drying to obtain a low-value fish hydrolysate dry powder.

[0016] Preferably, the composite protease comprises alkaline protease and neutral protease, and the mass ratio of the alkaline protease to the neutral protease is 1:1.

[0017] Further preferably, the enzyme activity of the alkaline protease is 2×10 5 U / g, the enzyme activity of the neutral protease is 1×10 5 U / g, its activity unit definition and detection method refer to the national standard GB / T 23527-2009.

[0018] Preferably, the process parameters of the spray drying are: liquid temperature 50-55°C, inlet temperature 180-200°C, and outlet temperature 70-80°C.

[0019] Preferably, the method for preparing the fermentation broth of Bacillus velez NKBV-01 comprises the following steps: (1) Inoculate Bacillus velezensis NKBV-01 on a slant of LB solid culture medium and culture at 35-37°C for 20-24 hours to obtain an activated strain; (2) Inoculate the activated strain into LB liquid culture medium, and culture with shaking at 35-37°C and 200-220 rpm for 20-24 h to obtain seed solution; (3) Inoculate the seed liquid into LB liquid culture medium at a volume ratio of 1% to 3%, control the ventilation volume at 5 to 10 L / min, and culture under shaking conditions at 35 to 37°C and 200 to 220 rpm for 20 to 24 hours to obtain bacterial liquid; (4) Inoculate the bacterial liquid into the fermentation medium at a volume ratio of 2% to 5%, control the ventilation volume at 300 to 400 L / min, and culture at 35 to 37°C and 200 to 220 rpm for 48 to 72 h; remove the residue to obtain the Bacillus Velezii NKBV-01 fermentation liquid.

[0020] Preferably, the fermentation medium in step (4) comprises the following components: 130 g / L avermectin fermentation waste residue dry powder, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 0.25 g / L neutral protease, and the initial pH value is 6.5-7.5.

[0021] Further preferably, the enzyme activity of the neutral protease is 1×10 5 U / g, its activity unit definition and detection method refer to the national standard GB / T 23527-2009.

[0022] Preferably, the method for preparing the fermentation medium comprises the following steps: after the above components are formulated, enzymatic hydrolysis is performed at 50-55°C for 120-180 min; and then heating and sterilizing at 121°C for 20-30 min to obtain the fermentation medium.

[0023] Preferably, the content of live bacteria of Bacillus Velez NKBV-01 in the fermentation broth is (1.5-2.5)×10 10 cfu / mL.

[0024] Preferably, the live bacteria content of Bacillus Velezii NKBV-01 in the water-soluble microbial agent is ≥ 5×10 9 cfu / g, the content of water-soluble organic matter is ≥150g / L.

[0025] The water-soluble microbial agent is used in promoting the growth of tomato plants and harmless treatment of avermectin fermentation waste residue.

[0026] Preferably, the method for applying the water-soluble microbial agent in promoting the growth of tomato plants is: mixing the water-soluble microbial agent with water in a weight ratio of 1:200-1000 and then drip irrigation or flushing.

[0027] Preferably, the application method of the water-soluble microbial agent in the harmless treatment of avermectin fermentation waste residue is: after mixing the water-soluble microbial agent with water in a weight ratio of 1:10 to 20, spraying it on the surface of the avermectin fermentation waste residue compost, and stirring it thoroughly.

[0028] Beneficial effects of the present invention: (1) The present invention provides a strain of Bacillus Velezii NKBV-01, which has the functions of producing extracellular polysaccharides, promoting soil aggregate formation, degrading avermectin, hydrolyzing protein, producing indoleacetic acid, promoting tomato plant growth, and promoting the enzyme activity of tomato rhizosphere soil enzymes; a water-soluble microbial agent prepared based on the strain not only has the effect of improving soil aggregate structure and promoting tomato plant growth, but also can accelerate the temperature rise of avermectin fermentation waste residue composting, prolong the high temperature period and shorten the composting cycle.

[0029] (2) The present invention utilizes fermentation strains of avermectin fermentation waste residues and uses low-value fish hydrolysates as the main raw materials to prepare water-soluble microbial agents, which not only helps to reduce production costs and broaden the source of raw materials, but also helps to promote the harmless treatment and resource utilization of avermectin fermentation waste residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the colony morphology of strain NKBV-01.

[0031] Figure 2 This is the bacterial morphology of strain NKBV-01.

[0032] Figure 3 is the phylogenetic tree of strain NKBV-01.

[0033] Figure 4 This is the temperature change during the composting process in Application Example 2. DETAILED DESCRIPTION

[0034] The following is explained in conjunction with specific embodiments: Source of experimental materials: Low-value fish meal: purchased from Xindeli Biotechnology Co., Ltd., Tai'an City, Shandong Province, and is the waste material of producing feed fish meal.

[0035] Example 1: Isolation and screening of Bacillus velez NKBV-01 The samples were collected from the compost of avermectin fermentation waste residue in Ancheng Town, Pingyin County, Jinan City, Shandong Province, China. The specific separation and screening methods were as follows: 10 g of compost sample was weighed, 100 mL of sterile water was added, and the sample was placed on a constant temperature shaker at 35°C and 180 r / min for 30 min of shaking to obtain an extract.

[0036] (1) Screening of avermectin-degrading strains: The above-mentioned extract was inoculated into LB liquid culture medium containing 200 mg / L avermectin, and cultured at 35°C and 180 r / min for 3 days; the culture medium was then transferred to LB liquid culture medium containing 400 mg / L, 600 mg / L, and 800 mg / L avermectin in turn, and cultured for 3 days respectively to obtain enriched culture; the enriched culture was appropriately diluted, and then spread on LB solid culture medium with a concentration of 1 / 10 containing 800 mg / L avermectin, and cultured at 35°C until a single colony grew; the single colony was separated to obtain strains with different colony morphologies, and then inoculated into culture medium with avermectin as the sole carbon source, and cultured at 35°C and 180 r / min for 24 to 72 h. The strain that could make the culture medium turbid was selected, i.e., the strain that could degrade avermectin was obtained, and stored for later use.

[0037] The components of the LB liquid culture medium are: 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 1000 mL of distilled water; pH 7.0, sterilization at 121° C. for 20 min.

[0038] The LB solid medium with a concentration of 1 / 10 containing 800 mg / L avermectin is a medium in which the concentration of each nutrient is 1 / 10 of the conventional LB solid medium, and contains 800 mg / L avermectin; the specific components are: 1 g of peptone, 0.5 g of yeast extract, 1 g of NaCl, 800 mg of avermectin, 20 g of agar, and 1000 mL of distilled water.

[0039] The components of the culture medium with avermectin as the sole carbon source are: (NH4)2SO42g, MgSO40.2g, NaH2PO40.5g, CaCl20.1g, K2HPO40.5g, avermectin 800mg, and distilled water 1000mL.

[0040] (2) Screening of extracellular polysaccharide-producing strains: The strains that can degrade avermectin selected above were inoculated into the polysaccharide-producing fermentation medium and cultured at 35°C and 220 r / min for 48 h to obtain the fermentation broth; the fermentation broth was centrifuged at 5000 r / min for 15 min, the supernatant was taken, and 3 times the volume of 95% (v / v) ethanol solution of the supernatant was added. The mixture was mixed evenly and allowed to stand at 4°C for 2 h; if flocculent precipitates were produced, it could be determined that the strain was capable of producing extracellular polysaccharides, and the more flocculent precipitates, the stronger the strain's ability to produce extracellular polysaccharides.

[0041] The polysaccharide-producing fermentation medium has a formula of: NaNO3 0.5 g / L, K2HPO4 0.5 g / L, MgSO4 0.5 g / L, NaCl 10 g / L, sucrose 25 g / L, yeast extract 3 g / L, peptone 3 g / L, and pH 7.0-7.2.

[0042] Through the above isolation and screening work, we finally screened out a strain that has the characteristics of degrading avermectin and producing extracellular polysaccharides, and named it "NKBV-01".

[0043] Example 2: Identification of Bacillus velez NKBV-01 The colony morphology of the strain NKBV-01 obtained by the above screening on LB solid medium is as follows: Figure 1 As shown, the colonies are round, milky white, and opaque; the bacterial morphology of strain NKBV-01 is as follows Figure 2 As shown, the bacterial body is rod-shaped and has the ability to form spores.

[0044] In addition, the physiological and biochemical characteristics of strain NKBV-01 were identified, and the identification results were as follows: catalase test+; methyl red test+; starch hydrolysis test+; denitrification test+; ammonia production test+; lactose fermentation test-; sucrose fermentation test+; xylose fermentation test+; arabinose test+; salicin test-; melibiose fermentation test+; rhamnose fermentation test-; mannitol fermentation test+; glucose fermentation test+ (+, positive; -, negative).

[0045] The genomic DNA of strain NKBV-01 was extracted and PCR amplified using 16S rDNA universal primers. The amplification conditions were: 95℃ pre-denaturation for 3min; 94℃ denaturation for 1min, 55℃ renaturation for 1min, 72℃ extension for 1.5min, a total of 30 cycles; 72℃ extension for 10min. The amplified 16S rDNA gene sequence was sequenced, and the sequencing results are shown in SEQ ID NO.1; the obtained 16S rDNA sequence was compared with the existing sequences in the GenBank database, and multiple sequence homology analysis was performed to construct a phylogenetic tree. The results are shown in Figure 3 shown by Figure 3 It can be obtained that the strain NKBV-01 obtained by the above screening is Bacillus velezensis The strain psa-3 Bacteria is relatively close in evolutionary distance. Combined with the physiological and biochemical characteristics of the strain, it was identified as "Bacillus Velezii ( Bacillus velezensis )".

[0046] Bacillus Velez Bacillus velezensis ) NKBV-01, deposited on November 6, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32500.

[0047] Example 3: Determination of beneficial properties of Bacillus velez NKBV-01 (1) Exopolysaccharide production capacity of Bacillus velez NKBV-01 The strain NKBV-01 was inoculated into LB liquid culture medium, and cultured under shaking conditions of 35°C and 200 r / min for 16 hours to obtain seed liquid; the seed liquid was inoculated into polysaccharide fermentation medium at a volume ratio of 5%, and continued to culture under shaking conditions of 35°C and 200 r / min for 60 hours to obtain fermentation liquid; the fermentation liquid was centrifuged at 5000 r / min for 15 minutes, 10 mL of supernatant was taken, 40 mL of anhydrous ethanol was added, and after mixing evenly, it was allowed to stand at 4°C for 2 hours to precipitate polysaccharides; the centrifugation was continued at 5000 r / min for 15 minutes, the supernatant was removed, and the precipitate was dissolved with 4 mL of distilled water to obtain a polysaccharide crude extract, and the polysaccharide content was determined by the phenol-sulfuric acid method.

[0048] The experimental results show that the polysaccharide content in the above-mentioned polysaccharide crude extract reaches 1.62 g / L, which indicates that Bacillus Velezii NKBV-01 has a strong ability to produce extracellular polysaccharides.

[0049] (2) Effects of Bacillus velezensis NKBV-01 and its exopolysaccharides on soil aggregate formation The strain NKBV-01 was inoculated into LB liquid culture medium, and cultured at 35°C and 200 r / min for 16 hours to obtain seed liquid; the seed liquid was inoculated into polysaccharide fermentation medium at a volume ratio of 5%, and cultured at 35°C and 200 r / min for 60 hours to obtain fermentation liquid.

[0050] The fermentation liquid was divided into two parts, one of which was normal fermentation liquid, and the other was sterilized at 121℃ for 30min to obtain inactivated fermentation liquid. 20mL of polysaccharide-producing fermentation medium (control group), inactivated fermentation liquid and normal fermentation liquid were taken respectively and added into Erlenmeyer flasks containing 200g of soil (passed through a 0.25mm sieve), mixed evenly, and cultured at room temperature for 45d. During this period, deionized water was regularly added to keep the soil surface moist. Three replicates were set for each treatment group, and the proportion of water-stable aggregates (>0.25mm) in the soil was determined.

[0051] The results are shown in Table 1 below, where different letters represent significant differences (P < 0.05).

[0052] Table 1. Proportion of water-stable aggregates in different treatment groups

[0053] As shown in Table 1, compared with the control group, the proportion of water-stable aggregates in the soil of the inactivated fermentation liquid treatment group and the normal fermentation liquid treatment group increased by 33.06% and 62.33%, respectively; compared with the inactivated fermentation liquid treatment group, the proportion of water-stable aggregates in the soil of the normal fermentation liquid treatment group increased by 21.99%. The experimental results show that Bacillus Velezii NKBV-01 and its exopolysaccharides can significantly promote the formation of soil aggregates.

[0054] (3) Degradation ability of Bacillus velez NKBV-01 on avermectin The strain PAPM-LX25 was inoculated into the avermectin fermentation waste residue culture medium, and cultured at 35°C and 200r / min for 72h to obtain the culture solution. The initial concentration of avermectin in the avermectin fermentation waste residue culture medium and the residual avermectin concentration in the culture solution were determined by liquid chromatography technology, and the degradation rate of avermectin by the strain PAPM-LX25 was calculated.

[0055] The preparation method of the avermectin fermentation waste residue culture medium is as follows: after drying the avermectin fermentation waste residue, crush it to 200 mesh, weigh 10g of powder, add 100mL of distilled water, adjust the pH value to 6.5, and sterilize at 121°C for 15min. The avermectin fermentation waste residue is solid waste generated during the fermentation production process of avermectin, and its organic matter content is 75.86%, the nitrogen, phosphorus and potassium contents are 3.62%, 1.35% and 0.63% respectively, the avermectin residue is 2.857mg / g, and the moisture content is 4.82%.

[0056] The experimental results showed that the degradation rate of avermectin by Bacillus Velez PAPM-LX25 could reach 96.87%.

[0057] (4) Protein degradation ability of Bacillus velez NKBV-01 The strain NKBV-01 was inoculated into skim milk powder solid medium and cultured at 35°C for 48 hours. The diameter of the transparent circle and the diameter of the colony produced in the medium were measured, and the diameter ratio (HC) of the transparent circle to the colony was calculated. The results showed that the diameter of the transparent circle formed by the strain NKBV-01 on the medium was 18.7 mm, the diameter of the colony was 4.6 mm, and the HC value reached 4.07.

[0058] The components of the skimmed milk powder solid culture medium are as follows: 3.0 g skimmed milk powder, 1 g glucose, 3.2 g agar, 200 mL deionized water, pH 7.0, and high-temperature sterilization at 108° C. for 15 min.

[0059] The experimental results showed that Bacillus Velez PAPM-LX25 had excellent protein hydrolysis ability, which helped the strain to hydrolyze avermectin fermentation waste residue, to grow and reproduce in the soil, and to decompose and transform organic nutrients in the soil.

[0060] (5) Ability of Bacillus velezensis NKBV-01 to produce indoleacetic acid The strain NKBV-01 was inoculated into LB liquid medium containing 200 mg / L L-tryptophan, and cultured at 35°C and 200 r / min for 3 days to obtain a bacterial suspension; 50 μL of the bacterial suspension was dripped onto a white ceramic plate and mixed with 50 μL of Salkowski colorimetric solution; a Salkowski colorimetric solution containing 50 mg / L IAA was set as a positive control. The white ceramic plate was placed at room temperature in the dark for 30 minutes, and the color change was observed. If the color turned red, it indicated that the strain had the ability to secrete indoleacetic acid.

[0061] The components of the Salkowski colorimetric solution are as follows: 450 mL of 35% HClO solution and 1 mL of 0.5 mol / L FeCl3 solution.

[0062] The experimental results showed that the ceramic plate turned dark red after the NKBV-01 bacterial suspension was added, which indicated that the Bacillus Velezii NKBV-01 strain had excellent indoleacetic acid production ability.

[0063] Example 4: Preparation of water-soluble microbial agent 1. Preparation of low-value fish enzymatic hydrolysate dry powder Low-value fish meal is used as a substrate to prepare a slurry with a substrate mass percentage of 10%. After being stirred evenly, the slurry is heated at 85°C for 15 minutes to achieve moderate protein denaturation; after the slurry is cooled to below 55°C, a composite protease is added at a ratio of 2% of the substrate mass percentage, and then enzymolysis is carried out at 52.5°C for 5 hours; after the enzymolysis is completed, the temperature is raised to 85°C and maintained for 15 minutes to inactivate the enzyme, and then the temperature is lowered to below 55°C; solid-liquid separation is carried out through a plate and frame filter to obtain a clarified filtrate, the filtrate is concentrated to a solid mass percentage of 25%, and spray-dried to obtain a low-value fish enzymolysate dry powder.

[0064] The composite protease comprises alkaline protease and neutral protease, the mass ratio of the alkaline protease to the neutral protease is 1:1; the enzyme activity of the alkaline protease is 2×10 5 U / g, the enzyme activity of the neutral protease is 1×10 5 U / g, its activity unit definition and detection method refer to the national standard GB / T 23527-2009.

[0065] The process parameters of the spray drying are as follows: liquid temperature 50-55°C, inlet temperature 180-200°C, and outlet temperature 70-80°C.

[0066] 2. Preparation of Bacillus Velezii NKBV-01 fermentation broth (1) Inoculate Bacillus velezensis NKBV-01 on the slant of a test tube containing LB solid culture medium and culture at 37°C for 24 hours to obtain an activated strain; (2) Using an inoculation loop, inoculate the activated strain into a flask containing LB liquid culture medium, and culture with shaking at 37°C and 200 rpm for 24 h to obtain seed solution; (3) Inoculate the seed liquid into a 10L seed tank containing 8L LB liquid medium at a volume ratio of 3%, control the ventilation volume at 8L / min, and shake culture at 37°C and 220rpm for 20h to obtain the seed tank bacterial liquid; (4) The bacterial liquid in the seed tank was inoculated into a 1000L fermentation tank containing 700L fermentation medium at a volume ratio of 5%, and the ventilation volume was controlled at 350L / min. The culture was carried out at 35°C and 220rpm for 60h. After the culture was completed, the residue was removed by a 60-mesh vibrating screen, and the suspension that could pass through the vibrating screen was collected, i.e., the fermentation liquid of Bacillus Velezii NKBV-01, in which the viable bacterial content of strain NKBV-01 was 2.0×10 10 cfu / mL.

[0067] The components of the fermentation medium in step (4) are as follows: 130 g / L of avermectin fermentation waste powder, 0.5 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, and 0.25 g / L of neutral protease, with an initial pH value of 6.5-7.5; the enzyme activity of the neutral protease is 1×10 5 U / g, and the definition and detection method of the activity unit refer to the national standard GB / T 23527-2009. After the above components are prepared, the fermentation medium is obtained by enzymolysis at 50°C for 120 minutes and sterilization at 121°C for 20 minutes.

[0068] The preparation method of the avermectin fermentation waste residue dry powder is as follows: drying the avermectin fermentation waste residue and crushing it to 200 meshes. The avermectin fermentation waste residue is solid waste generated during the avermectin fermentation production process, and its organic matter content is 75.86%, the nitrogen, phosphorus and potassium contents are 3.62%, 1.35% and 0.63% respectively, the avermectin residue is 2.857 mg / g, and the moisture content is 4.82%.

[0069] 3. Preparation of water-soluble microbial agents Take 80 parts by weight of Bacillus Velez NKBV-01 fermentation liquid, 19.9 parts by weight of low-value fish enzymolyte dry powder, and 0.1 parts by weight of kason; slowly add the low-value fish enzymolyte dry powder and kason into the Bacillus Velez NKBV-01 fermentation liquid, and continue stirring until the mixture is evenly mixed and no block particles are visible, to obtain a water-soluble microbial agent.

[0070] Wherein, the live bacteria content of Bacillus Velez NKBV-01 in the water-soluble microbial agent is ≥5×10 9 cfu / g, the content of water-soluble organic matter is ≥150g / L.

[0071] Application Example 1: Application of water-soluble microbial agents in promoting the growth of tomato plants This experiment was carried out in Ancheng Town, Pingyin County, Shandong Province. The specifications of the flower pots used were 20 cm in upper inner diameter, 14 cm in lower inner diameter, and 16 cm in height. Each pot was filled with 3 kg of soil, and the crops planted in the pots were tomatoes.

[0072] Treatment group T1, treatment group T2 and control group CK were set up, and 20 repetitions were set up in each group. Among them: 15 mL of the fermentation liquid of Bacillus Velez NKBV-01 prepared in Example 4 was applied to each pot of soil in treatment group T1; 15 mL of the water-soluble microbial agent prepared in Example 4 was applied to each pot of soil in treatment group T2; 15 mL of the fermentation medium described in Example 4 was applied to each pot of soil in control group CK. The application method was flushing with water, and the application amount each time was 5 mL, which was diluted 200 times with water according to the weight ratio and then slowly poured into the flower pot; the first application time was the 7th day after tomato transplanting, and then it was applied every 10 days, for a total of 3 times.

[0073] At 60 days after tomato transplanting, the plant height, stem diameter, fresh weight, rhizosphere soil enzyme activity and other related indicators of tomato plants were measured. Among them, plant height and stem diameter were measured by tape measure and vernier caliper respectively; fresh weight was measured by weighing method; rhizosphere soil enzyme activity was measured by the following methods: urease activity was measured by sodium phenol colorimetry, catalase activity was measured by potassium permanganate titration method, and sucrase activity was measured by DNS colorimetry.

[0074] The test results are shown in Tables 2-3, where different letters represent significant differences (P < 0.05).

[0075] Table 2. Effects of water-soluble microbial agents on the growth of tomato plants

[0076] It can be seen from Table 2 that compared with the control group CK, the treatment groups T1 and T2 showed different degrees of improvement in the plant height, stem diameter and fresh weight of tomato plants, which indicates that Bacillus Velez NKBV-01 can promote the growth of tomato plants; compared with the treatment group T1, the treatment group T2 had an increase rate of 16.18% in the plant height, 20.00% in the stem diameter, and 21.94% in the fresh weight.

[0077] It can be seen that the water-soluble microbial agent prepared with Bacillus Velez NKBV-01 as the main ingredient has a significant promoting effect on the growth of tomato plants.

[0078] Table 3. Effects of water-soluble microbial agents on enzyme activities in tomato rhizosphere soil

[0079] It can be seen from Table 3 that compared with the control group CK, the activities of urease, catalase and sucrase in the rhizosphere soil of tomato in treatment groups T1 and T2 were significantly improved, which indicates that Bacillus Velezii NKBV-01 can significantly promote the enzyme activity of tomato rhizosphere soil enzymes; compared with treatment group T1, the increase rate of urease activity in the rhizosphere soil of tomato in treatment group T2 was 18.12%, the increase rate of catalase activity was 13.33%, and the increase rate of sucrase activity was 6.23%.

[0080] It can be seen that the water-soluble microbial agent prepared with Bacillus Velez NKBV-01 as the main ingredient can significantly promote the enzyme activity of tomato rhizosphere soil enzymes.

[0081] Application Example 2: Application of water-soluble microbial agents in harmless treatment of avermectin fermentation waste residue This experiment was carried out at Shandong Zuotianshi Biotechnology Co., Ltd. (Ancheng Town, Pingyin County, Shandong Province). The composting container used was a foam box with an internal dimension of 60 cm in length, 45 cm in width, 41 cm in height, and a wall thickness of 5 cm. The composting raw materials consisted of 25% avermectin fermentation waste residue and 75% edible fungus bran in terms of mass ratio. The loading capacity of each box was 40 kg, and the initial moisture content was 55%.

[0082] A treatment group T, a control group CK1 and a control group CK2 were set, wherein: the treatment group T was inoculated with the water-soluble microbial agent prepared in Example 4, the control group CK1 was not inoculated with any agent, and the control group CK2 was inoculated with a commercially available organic material decomposition agent, and the inoculation amount of each group was 0.1% (mass percentage). The inoculation method was to add the inoculum to 20 times the weight of water, stir evenly, spray it on the surface of the compost, and stir it thoroughly. Within 50 days after composting, the compost was turned every 1 to 2 days, and then every 5 days, in order to supply oxygen and maintain the uniform state of the compost raw materials.

[0083] Among them, the avermectin fermentation waste residue is the solid waste generated in the avermectin fermentation production process, with an organic matter content of 75.86%, nitrogen, phosphorus and potassium contents of 3.62%, 1.35% and 0.63% respectively, an avermectin residue of 2.857 mg / g, and a moisture content of 4.82%.

[0084] Among them, the edible fungus bran is a by-product of the cultivation process of Flammulina velutipes, and its organic matter content is 71.35%, and the contents of nitrogen, phosphorus and potassium are 1.52%, 2.38% and 1.21% respectively (all based on dry matter), and the moisture content is 56.75%.

[0085] Among them, the commercially available organic material composting agent is produced by Shandong Zuotianshi Biotechnology Co., Ltd., and its effective live bacteria count is 1 billion cfu / g. The bacteria in the composting agent include Bacillus subtilis, Bacillus licheniformis, Aspergillus niger and yeast.

[0086] The test results are as follows Figure 4 As shown. Figure 4 It can be seen that the compost temperature of the control group CK1, CK2 and the treatment group T reached 50°C on the 10th day, the 10th day and the 4th day, respectively, and the high temperature duration was 10 days, 10 days and 14 days, respectively. This shows that the water-soluble microbial agent provided by the present invention can significantly accelerate the decomposition process of avermectin fermentation waste residue, prolong the high temperature period and shorten the composting cycle, which has positive significance for the harmless treatment of avermectin fermentation waste residue, improving the composting efficiency of avermectin fermentation waste residue and improving product quality.

Claims

1. A strain of Bacillus velez Bacillus velezensis ) NKBV-01, deposited on November 6, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32500.

2. The use of the Bacillus Velez NKBV-01 of claim 1 in producing exopolysaccharides, promoting soil aggregate formation, degrading avermectin, hydrolyzing protein, producing indoleacetic acid, promoting tomato plant growth, and promoting enzyme activity of tomato rhizosphere soil enzymes; Preferably, the tomato rhizosphere soil enzymes include urease, catalase and sucrase.

3. A water-soluble microbial agent, characterized in that: The method comprises the Bacillus Velezii NKBV-01 according to claim 1; Preferably, the water-soluble microbial agent also includes low-value fish enzymatic hydrolysate dry powder and kason; Further preferably, the water-soluble microbial agent comprises the following components: 75-85 parts by weight of Bacillus Velez NKBV-01 fermentation broth, 14.85-24.95 parts by weight of low-value fish enzymatic hydrolysate dry powder, and 0.05-0.15 parts by weight of kason; More preferably, the water-soluble microbial agent comprises the following components: 80 parts by weight of Bacillus Velez NKBV-01 fermentation broth, 19.9 parts by weight of low-value fish enzymatic hydrolysate dry powder, and 0.1 parts by weight of Kathon.

4. The water-soluble microbial agent according to claim 3, characterized in that The live bacteria content of Bacillus Velezii NKBV-01 is ≥5×10 9 cfu / g, the content of water-soluble organic matter is ≥150g / L.

5. The method for preparing the water-soluble microbial agent according to claim 3, characterized in that: The method comprises the following steps: adding low-value fish enzymatic hydrolysate dry powder and cassonne into the fermentation liquid of Bacillus Velez NKBV-01, mixing evenly, and obtaining a water-soluble microbial agent.

6. The preparation method according to claim 5, characterized in that: The preparation method of the low-value fish enzymolyte dry powder comprises the following steps: using low-value fish meal as a substrate, preparing a feed liquid with a substrate mass percentage of 8% to 12%, heating the feed liquid at 80 to 90° C. for 10 to 20 minutes to achieve protein denaturation; after the feed liquid is cooled, adding a composite protease at a ratio of 1% to 3% of the substrate mass percentage, and then performing enzymolysis at 50 to 55° C. for 3 to 5 hours; after the enzymolysis is completed, heating to 80 to 90° C. and maintaining for 10 to 20 minutes to inactivate the enzyme, and then reducing the temperature to below 55° C.; performing solid-liquid separation to obtain a filtrate, concentrating the filtrate to a solid mass percentage of ≥25%, and spray drying to obtain a low-value fish enzymolyte dry powder; Preferably, the composite protease comprises alkaline protease and neutral protease, and the mass ratio of the alkaline protease to the neutral protease is 1:1; Further preferably, the enzyme activity of the alkaline protease is 2×10 5 U / g, the enzyme activity of the neutral protease is 1×10 5 U / g, its activity unit definition and detection method refer to the national standard GB / T 23527-2009; Preferably, the process parameters of the spray drying are: liquid temperature 50-55°C, inlet temperature 180-200°C, and outlet temperature 70-80°C.

7. The preparation method according to claim 5, characterized in that: The preparation method of the Bacillus Velez NKBV-01 fermentation liquid comprises the following steps: (1) Inoculate Bacillus velezensis NKBV-01 on a slant of LB solid culture medium and culture at 35-37°C for 20-24 hours to obtain an activated strain; (2) Inoculate the activated strain into LB liquid culture medium, and culture with shaking at 35-37°C and 200-220 rpm for 20-24 h to obtain seed solution; (3) Inoculate the seed liquid into LB liquid culture medium at a volume ratio of 1% to 3%, control the ventilation volume at 5 to 10 L / min, and culture under shaking conditions at 35 to 37°C and 200 to 220 rpm for 20 to 24 hours to obtain bacterial liquid; (4) Inoculate the bacterial liquid into the fermentation medium at a volume ratio of 2% to 5%, control the ventilation volume at 300 to 400 L / min, and culture at 35 to 37°C and 200 to 220 rpm for 48 to 72 hours; remove the residue to obtain the fermentation liquid of Bacillus Velezii NKBV-01; Preferably, the fermentation medium in step (4) comprises the following components: 130 g / L of avermectin fermentation waste residue dry powder, 0.5 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, and 0.25 g / L of neutral protease, and the initial pH value is 6.5-7.5; Preferably, the enzyme activity of the neutral protease is 1×10 5 U / g, its activity unit definition and detection method refer to the national standard GB / T 23527-2009.

8. The preparation method according to claim 5, characterized in that: The content of live bacteria of Bacillus Velez NKBV-01 in the fermentation liquid of Bacillus Velez NKBV-01 is (1.5~2.5)×10 10 cfu / mL.

9. Use of the water-soluble microbial agent according to claim 3 in promoting the growth of tomato plants and harmless treatment of avermectin fermentation waste residue.

10. The use according to claim 9, characterized in that The method for applying the water-soluble microbial agent to promote the growth of tomato plants is as follows: the water-soluble microbial agent is mixed with water in a weight ratio of 1:200-1000 and then drip-irrigated or flushed; The application method of the water-soluble microbial agent in the harmless treatment of avermectin fermentation waste residue is: after mixing the water-soluble microbial agent with water in a weight ratio of 1:10-20, spraying it on the surface of the avermectin fermentation waste residue compost and stirring it thoroughly.

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